Methods and compositions for tailing and amplifying RNA

Abstract

The present invention provides methods and compositions that enable tagging and amplification of targeted RNA molecules. A targeted RNA molecule is any non-polyadenylated RNA molecule including, for example, miRNA, siRNA, rRNA, tRNA, synthetic RNA, or non-polyadenylated mRNA such as mRNA from bacteria. In certain aspects, the invention provides methods and compositions for the genome-wide expression analysis of bacterial genes. Significantly, the methods enable genome-wide expression analysis in circumstances where bacterial numbers were previously too low to purify adequate amounts of RNA for DNA microarray analysis or other applications. Such methods are particularly useful for the study of bacterial gene expression during host-cell infection. The invention also provides kits for tagging and amplifying targeted RNA molecules.

Description

BACKGROUND OF THE INVENTION [0001] 1. Field of the Invention [0002] The present invention relates generally to the field of molecular biology. More particularly, it concerns methods and compositions for tailing and amplifying non-polyadenylated RNA molecules, including microRNA, siRNA, tRNA, rRNA, synthetic RNA, and non-polyadenylated mRNA, such as mRNA from bacteria or fragments of prokaryotic or eukaryotic mRNAs such as may be encountered in degraded RNA samples. [0003] 2. Description of Related Art [0004] The commonly used method for mRNA purification from eukaryotic cells, oligo-dT capture, is ineffective for capturing RNA molecules lacking poly(A) tails. Examples of RNA molecules that are not polyadenylated include microRNA, siRNA, tRNA, rRNA, synthetic RNA, and non-polyadenylated mRNA from prokaryotes. In addition, mRNA fragments, such as may be encountered in degraded RNA samples, may also lack poly(A) tails. The study of these important RNA molecules is hindered by inadequat...

AI Technical Summary

Benefits of technology

[0016] The present invention provides methods and compositions for tailing and amplifying a targeted RNA molecule. A targeted RNA molecule may be any non-polyadenylated RNA molecule. Examples of non-polyadenylated RNA include microRNA, siRNA, tRNA, rRNA, synthetic RNA, or non-polyadenylated mRNA, such as mRNA from bacteria or fragments of prokaryotic or eukaryotic mRNAs such as may be encountered in degraded RNA samples. Advantages of the present invention include the ability to efficiently and uniformly tail a targeted RNA and the ability to representatively amplify a population of targeted RNA molecules in a sample.

[0018] In one embodiment, the invention provides a method for increasing the efficiency of tailing a targeted RNA in a sample comprising altering the secondary structure of the targeted RNA, and incubating the targeted RNA in the presence of an enzyme that adds a nucleic acid tail under conditions that allow tailing of the targeted RNA. As used herein, “increasing the efficiency of tailing” refers to increasing the percentage of targeted RNA molecules that are tailed during the reaction. In preferred embodiments, at least about 75%, 80%, 85%, or 90% of the targeted RNA molecules in a sample are tailed. More preferably, at least about 95% of the targeted RNA molecules are tailed. Even more preferably, at least about 99% of the targeted RNA molecules are tailed. In certain embodiments, about 95%, 96%, 97%, 98%, 99%, or about 100% of the targeted RNA molecules are tailed.

[0044] Depleting rRNA from the sample can enable the relative enrichment of targeted RNA to total RNA in a sample by about or at least about 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 fold or any range derivable therein.

[0053] In certain aspects of the invention, the targeted RNA and / or amplified RNA are labeled. Labeling the targeted RNA and / or amplified RNA facilitates the detection of the molecules in applications such as expression analysis. A number of different labels may be used in the present invention such as fluorophores, chromophores, radiophores, enzymatic tags, antibodies, chemiluminescence, electroluminescence, and affinity labels. Those of skill in the art are familiar with methods for labeling nucleic acids and will recognize that these and other labels not mentioned herein can be used with success in this invention.

Problems solved by technology

The commonly used method for mRNA purification from eukaryotic cells, oligo-dT capture, is ineffective for capturing RNA molecules lacking poly(A) tails.

The study of these important RNA molecules is hindered by inadequate methods for their isolation and amplification.

Another example of the difficulties encountered when analyzing non-polydenylated RNA molecules is the study of bacterial gene expression.

However, methods for in vivo whole genome expression analyses have not been fully realized.

The technical difficulties that hinder these studies include (1) purifying bacterial RNA free from large amounts of contaminating host cell RNA, and (2) purifying adequate amounts of bacterial RNA for DNA microarray analysis.

Identifying bacterial genes whose expression is altered by host cells has not been easy, and microbiologists have used numerous techniques to address the challenge.

However, all of these methods have limitations.

Most require significant genetic manipulation (construction of mutants, libraries, gene fusions) and are labor intensive.

Chief amongst the limitations is that none allows for quantification of genome-wide expression.

However, at this point, it too is limited by resolution and sensitivity.

If it is not, detergent treatment will likely alter the gene expression profile of the bacteria.

In addition, selective detergent lysis of host cells is less effective with many Gram-negative bacteria that are easily lysed with such detergents (e.g., Yersinia enterocolitica).

Furthermore, variable susceptibility of eukaryotic cells to saponin-mediated lysis makes optimization difficult.

But, this results in the loss of ˜30% of hybridization signals on E. coli arrays (Khil and Camerini-Otero, 2002; Arfin et al., 2000).

A small percentage of bacterial mRNAs are poly(A)-tailed, but these are targeted for degradation and tend to be unstable.

As a result, the commonly used method for mRNA purification with eukaryotic cells, oligo-dT capture, is ineffective for capturing bacterial mRNAs.

However, using their method Amara and Vijaya were not able to efficiently poly(A) tail the transcripts.

Optimizing the reaction to work reproducibly in many different bacterial cell lysates would likely be very difficult.

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